Evaluation of a new, rapid collagen assay.

Collagen quantitation is usually based on the measurement of its hydroxyproline content by chromatographic or by chemical methods [I]. Alternatively the uptake of radiolabelled proline may be used to monitor collagen production [2]. The chemical assay of hydroxyproline although sensitive is time consuming, requiring prior acid hydrolysis of the protein [ l ] . A fast and simple collagen assay has recently been developed, the Sircol Collagen Assay (Qubis Ltd., Belfast, BT9 5BN ). The assay is based on the specific binding by collagen of a dye reagent that is free from interference from non-collagenous proteins including albumin, elastin and FCS present in the test samples. The assay allows both salt soluble and acid soluble collagens to be assayed within the same test sample. The assay kit comprises four components; the Sircol dye reagent, an alkali reagent, a collagen standard and a salt soluble collagen precipitating reagent. We have evaluated this assay using a range of collagen samples including collagen, Type I from human placenta, calf skin and rat tail (Sigma), collagen, Type II from human hyaline cartilage (Novabiochem U.K. Ltd.) and collagen, Types 111, IV and V from human placenta (Sigma). An invertebrate source of collagen from the leech, Hirudo medicinalis (Biopharm U.K. Ltd.) was also assayed. The selective specificity of the binding between collagen and the dye reagent was examined in the presence of other proteins, including BSA, FCS and elastin . Tissue culture medium, RPMl 1640 (Gibco Ltd.. Paisley) was also examined in the presence and absence of FCS. Test samples were incubated with the Sircol dye reagent for 30 mins. after which they were centrifuged and the collagen bound dye eluted with the alkali reagent. The absorbance of the eluted dye was measured at 540nm. The intensity of the colour at 540nm was found to be proportional to the collagen concentration in the sample. _-----_______-____-________________